This invention is related to adrenoleukodystrophy. In particular, it is related to identification of mechanisms for overcoming the biochemical defects of this disease.
Functional gene redundancy and/or redundant metabolic pathways may be a fundamental aspect of vertebrate evolution. The observation that null mutations in mouse models of human disease created by targeted gene disruption often have no relevant clinical phenotype or a more subtle one than predicted has been explained in part by genetic redundancy. Redundant genes that can completely or partially substitute for each other are candidates for an approach to gene therapy for genetic disease that is based on increased expression of an endogenous gene rather than the introduction of a normal copy of the defective gene by transgenesis. This approach avoids the complications inherent in transgene gene therapy, such as targeting expression to the appropriate tissue and the immunosurveillance of tissues expressing the new transgene. In principle, enhanced gene expression could be accomplished in utero, allowing for early intervention. Initial exploration of pharmacologic induction of redundant genes in a clinical setting involved the stimulation of fetal hemoglobin by 5-azacytidine, hydroxyurea, sodium butyrate and sodium 4-phenylbutyrate to ameliorate the effects of adult hemoglobinopathies. Such an approach might be useful in Duchenne muscular dystrophy by increasing the expression of utrophin, a protein that is structurally similar to dystrophin, the abnormal protein in this genetic disorder. As more functional gene redundancy is recognized, increased expression of a related gene could become a general approach to the treatment of genetic disease. X-linked adrenoleukodystrophy (X-ALD; Mendelian Inheritance in Man number 300100) is a candidate for such pharmacological gene therapy.
X-ALD is associated with defective peroxisomal xcex2-oxidation of saturated very-long-chain fatty acids (VLCFA) and reduced activity of peroxisomal VLCF-acyl CoA synthetase. It affects mainly central and peripheral myelin, the adrenal cortex and the testis12. X-ALD shows a highly variable clinical phenotype including a rapidly progressive childhood cerebral form (CCER), with inflammatory cerebral demyelination; a milder adult form, adrenomyeloneuropathy (AMN) that is slowly regressive and with initial symptoms limited to the spinal cord and peripheral nerves of the limbs; and a form (Addison-only) in which there is adrenal insufficiency without neurologic involvement. All forms of X-ALD segregate in the same families and arise from identical mutations-including null mutations.
The gene for X-ALD, identified by positional cloning, encodes a peroxisomal membrane protein (ALDP) with a predicted molecular mass of 83 kDa. Based on sequence homology, it belongs to the ATP-binding cassette (ABC) superfamily of transmembrane transporters, with the structure of a half-transporter. Although peroxisomal VLCF-acyl CoA synthetase activity is impaired in X-ALD, mutational analysis- and complementation studies20,21 have shown that the gene for ALDP and not that for VLCF-acyl CoA synthetase is responsible for X-ALD.
There are three additional mammalian peroxisomal membrane ABC half-transporters that are closely related by nucleic acid and protein sequence: ALDRP (ALDPL1), an ALDP related protein; PMP70, a 70-kDa protein; and PMP69 (P70R), a 69-kDa protein. The function(s) of the peroxisomal ABC half-transporters and their interaction with VLCF-acyl CoA synthetase is unknown, but their considerable sequence similarity indicates that they might have related and/or overlapping function(s) in peroxisomal fatty acid metabolism. This is supported by the observations that X-ALD cells lacking ALDP have a residual activity for VLCFA xcex2-oxidation, which could result from one or more of the other peroxisomal ABC half-transporters; and that PMP70 overexpression partially restores VLCFA xcex2-oxidation in X-ALD fibroblasts, indicating that other peroxisomal ABC half-transporters can substitute, at least in part, for the absence of ALDP. In addition, the high level of identity between ALDP and ALDRP has led to speculation that these proteins might be functionally related.23 
We have generated a mouse model for X-ALD by targeted gene disruption29. The X-ALD mouse has elevated levels of VLCFA in tissues that resemble the characteristic biochemical defect in X-ALD patients. Thus, the in vivo efficacy of treatment can be monitored by determination of its effect on VLCFA levels in the tissues, brain and adrenal gland, the organs most affected in X-ALD.
At present, no completely satisfactory therapy for X-ALD is available. Some success has been achieved with bone marrow transplantation30,31 Lorenzo oil, a dietary therapy, depresses plasma and liver levels of VLCFA within a month; however, it has no effect on the clinical course of the disease, perhaps because erucic acid, the active ingredient of Lorenzo oil, does not get into the brain12,11. Thus, new therapy for X-ALD is needed.
4-Phenyl butyrate (4-PBA) has been used for many years in the treatment of patients with urea cycle disorders34 with few, if any, side effects. Although details of their mode of action are unclear, 4-PBA and other butyrate derivatives seem to increase expression of certain target genes35-38. Fenofibrate, a compound structurally related to 4-PBA, also increases expression of ALDRP and PMP70, but not ALDP, in rats39. Treatment of nasal epithelia cells from cystic fibrosis patients who are homozygous or heterozygous for the xcex94F508-CFTR mutation with 4-PBA results in rescue of xcex94F508-CFTR from premature degradation in the endoplasmatic reticulum. It is now believed that xcex94F508-CFTR protein biosynthesis is enhanced by 4-PBA because of altered regulation of protein folding by a chaperone in the endoplasmic reticulum. In X-ALD, 70% of mutations result in unstable ALDP. Thus, 4-PBA could similarly rescue unstable ALDP from premature degradation. In rodents, exposure to compounds related to 4-PBA, like clofibrate and fenofibrate, induces peroxisome proliferation44.
It is an object of the present invention to provide methods for treating patients with adrenoleukodystrophy.
It is another object of the present invention to provide methods of screening test substances to identify candidate therapeutic agents for treating adrenoleukodystrophy.
These and other objects of the invention are achieved by one or more of the embodiments described below. In one embodiment a method is provided of treating a patient with adrenoleukodystrophy. An effective amount of an agent which causes peroxisome proliferation is administered to a patient with adrenoleukodystrophy. As a result, the level of C24:0 or C26:0 fatty acids in the central nervous system of the patient is reduced.
According to another embodiment of the invention, a method is provided for treating a patient with adrenoleukodystrophy. An effective amount of an agent which increases the activity of a peroxisomal ATP binding cassette transmembrane transporter protein in the central nervous system of the patient is administered to the patient with adrenoleukodystrophy. As a result, the level of C24:0 or C26:0 fatty acids in the central nervous system of the patient is reduced.
Another embodiment of the invention provides a method of treating a patient with adrenoleukodystrophy. An effective amount of an agent which increases beta-oxidation of C24:0 or C26:0 fatty acids in the central nervous system of the patient is administered. As a result, the level of C24:0 or C26:0 fatty acids in the central nervous system of the patient is reduced.
Also provided by the present invention are methods of screening for candidate therapeutic agents for treating adrenoleukodystrophy. In each of the methods human cells are contacted with a test substance. In one method expression of ALDRP in the human cells is measured, and a test substance which increases expression of ALDRP is identified as a candidate therapeutic agent. In another method xcex2-oxidation of C24:0 or C26:0 fatty acids in the cells is measured, and a test substance which increases xcex2-oxidation is identified as a candidate therapeutic agent. In yet another of the methods peroxisome number in the cells is measured, and a test substance which increases peroxisome number in the cells is identified as a candidate therapeutic agent. In still another of the methods Pex11xcex1 in the cells, and a test substance which increases Pex11xcex1 in the cells is identified as a candidate therapeutic agent.
These and other embodiments of the invention which will be described in more detail below, and which will be evident to those of ordinary skill in the art upon reading the disclosure, provide the art with therapeutic methods for treating a metabolic disease and analytic methods for identifying additional therapeutic agents which have similar modes of action.